Transgenerational exposure to marine heatwaves ameliorates the lethal effect on tropical copepods regardless of predation stress

Abstract Marine heatwaves (MHWs) are emerging as a severe stressor in marine ecosystems. Extreme warm sea surface temperatures during MHWs often exceed the optimal thermal range for more than one generation of tropical coastal zooplankton. However, it is relatively unknown whether transgenerational plasticity (TGP) to MHWs may shape the offspring's fitness, particularly in an ecologically relevant context with biotic interactions such as predation stress. We addressed these novel research questions by determining the survival, reproductive success, and grazing rate of the copepod Pseudodiaptomus incisus exposed to MHW and fish predator cues (FPC) for two generations (F1 and F2). The experiment was designed in a full orthogonal manner with 4 treatments in F1 and 16 treatments in F2 generation. In both generations, MHW reduced P. incisus survival, reproductive parameters, and grazing by 10%–62% in MHW, but these parameters increased by 2%–15% with exposure to FPC, particularly at control temperature. F2 reproductive success and grazing rate as indicated by cumulative fecal pellets were reduced by 20%–30% in F1‐MHW, but increased by ~2% in F1‐FPC. Strikingly, MHW exposure reduced 17%–18% survival, but transgenerational exposure to MHWs fully ameliorated its lethal effect and this transgenerational effect was independent of FPC. Increased survival came with a cost of reduced reproductive success, constrained by reduced grazing. The rapid transgenerational MHW acclimation and its associated costs are likely widespread and crucial mechanisms underlying the resilience of coastal tropical zooplankton to MHWs in tropical coastal marine ecosystems.


| INTRODUC TI ON
Marine heatwaves (MHWs) are discrete periods of abnormally high temperatures (≥5 days) above the 90th percentile of 30-year sea surface temperatures measured at the locality (Hobday et al., 2016;Oliver et al., 2018). In the last decade, MHWs have emerged as a major driver reshaping marine biodiversity, causing the tropicalization of temperate coastal ecosystems, mass coral bleaching, and mass mortality of coastal invertebrates worldwide (Garrabou et al., 2009(Garrabou et al., , 2019Hughes et al., 2017;Smale et al., 2019). The effects of MHWs on marine ecosystems and biota are becoming more severe under ongoing climate change (Smale et al., 2019). In the Southeast Asian region, coastal marine organisms are increasingly being exposed to episodes of MHWs (Feng et al., 2022;Yao et al., 2020). The average duration of MHWs in the Southeast Asian seas is 18-22 days per event (Yao & Wang, 2021), which is equivalent to ~2-3 generations of tropical coastal invertebrates. A few recent studies have found strong negative effects of MHWs on marine organisms such as copepods and fish in the Southeast Asian region (e.g., Doan et al., 2019;Le et al., 2020;Nguyen, Le, Doan, Pham, et al., 2020), but these were mostly limited to one-exposed generation. It remains to be explored how transgenerational plasticity to MHWs might ameliorate the effects of warming on offspring generation (see, e.g., in coral fish, Donelson et al., 2012).
Advancements in eco-evolutionary studies on adaptations of organisms to toxic algae, warming, ocean acidification, and contaminants have further explored the critical role of transgenerational plasticity (TGP) where the environment experienced by the parental generation may improve offspring performance in the same environment (Colin & Dam, 2005;Donelson et al., 2012;Donelson et al., 2018;Fox et al., 2019;Guillaume et al., 2016;Krause et al., 2017;Munday, 2014;Thor & Dupont, 2015). For example, the tropical coral damselfish A. polyacanthus reduced the aerobic scope by 15-30% under acute exposure to an elevated temperature of +1.5°C and + 3°C, but the offspring's aerobic scope fully recovered when reared at the same temperature (Donelson et al., 2012). TGP is especially crucial for organisms to cope with new, predictable but fast-changing and short-term environmental changes across generations . The duration of an MHW often lasts longer than one generation for nearly all tropical zooplankton species. However, the duration of an MHW is often not long enough for directional evolutionary adaptation, as occurs in response to seasonal changes in water temperature (Sasaki & Dam, 2020) or long-term warming (Dam et al., 2021;Dinh Van et al., 2013). TGP generally occurs through epigenetic changes, habitat selection, or niche construction (reviewed in Donelan et al., 2020). In particular, epigenetic changes such as the methylation of genes encoding oxygen consumption, mitochondrial activity, and energy homeostasis play crucial functions in restoring the performance of stress-exposed organisms across generations (Ryu et al., 2018). Besides TGP, the parental effect is another non-genetic transgenerational effect, as the transfer of nutrients from mothers to eggs may also affect offspring performance (reviewed in Ho & Burggren, 2010). Parental effects of warming may negatively affect offspring performance, which has been shown in mosquito larvae with increased mortality and delayed development . Alternatively, genetic selection may occur through stressor-induced mortality (Krause et al., 2017) or selection in favor of higher hatching success (Dam et al., 2021), which removes the most sensitive genotypes or increases the tolerant genotypes in the population, and may contribute to increased fitness of the offspring generation.
In the shallow tropical coastal ecosystems such as seagrasses, mangroves, and coral reefs, the predation stress is typically high as these ecosystems are the spawning and nursery ground of marine species. Non-consumptive predation stress from voracious fish larvae and juveniles can significantly influence prey morphology, behavior, physiology, growth, and reproduction (e.g., Bjaerke et al., 2014;Lasley-Rasher & Yen, 2012;Truong et al., 2020). For example, the escape ability of copepods Notodiaptomus conifer and Argyrodiaptomus falcifer can be enhanced (Gutiérrez et al., 2010).
Calanoid copepods commonly show an increase in growth (e.g., Temora longicornis, Bjaerke et al., 2014) and reproduction (e.g., P. incisus, Truong et al., 2020) as general life-history trait responses to fish kairomones. Parental exposure to predators may also induce an increase in the reproduction of offspring generation (e.g., Daphnia magna) and this effect may last two generations after exposure to predators (Walsh et al., 2015). However, the TGP of prey species to predation stress may reduce in the degree of plasticity with an increasing number of exposed generations exposed to predators.
For example, the pea aphid (Acyrthosiphon pisum) responds to the presence of the predator ladybirds (Harmonia axyridis) by producing a higher frequency of the winged offspring, but this TGP response decreases across 22 exposed generations (Sentis et al., 2018).
Investigations of the transgenerational effect of MHWs in an ecologically relevant context, such as the presence of fish predator cues (FPC) on key zooplankton species, are relevant and timely with the increasing frequency, severity, and duration of MHWs and the intense predation stress of tropical coastal environments. Understanding whether copepods are resilient or vulnerable to MHWs in the context of predation stress is important, given that they are a key pathway for the transfer of energy and resources from photosynthesizing organisms to higher trophic levels, and ultimately the productivity of the coastal ecosystems (Chew et al., 2012). However, the combined effect of heatwaves and non-consumptive predation risk on prey species across generations is still a major knowledge gap in current ecological research. Our previous study shows that FPC induced a higher individual performance of the calanoid copepod Pseudodiaptomus incisus under control temperature, but it magnified the deleterious impacts of MHW on grazing and reproductive success . In this study, we address the knowledge gap identified above by assessing the immediate effect during the exposure (Dinh et al., 2016) together with the effects of parental exposure, TGP (parental and offspring exposure) to MHW, FPC, and their interactions in a full orthogonal manner with 4 treatments in F1 and 16 treatments in F2 generation. We tested the susceptibility of P. incisus to MHW and FPC by following eight hypotheses: H1 MHW reduces the performance of P. incisus due to energetic constraints under extreme warming .
H3 FPC-induced increase in performance of P. incisus is not sustained under MHW due to the energetic constraints .
H4 Parental exposure to MHW reduces offspring performance in the control temperature due to poor maternal provisioning .
H5 Parental exposure to FPC increases offspring performance in the absence of FPC (Walsh et al., 2015).
H8 The magnitude of TGP to MHW is altered by TGP to FPC, and vice versa. This is predicted based on the different types of responses of P. incisus to TGP to MHW and FPC.
We used a zooplankton net (mesh size = 200 μm) to collect Pseudodiaptomus incisus from a coastal aquaculture pond Healthy adult copepods (F0) were sorted and then divided into 5-L bottles, approximately 1200 individuals per bottle, for acclimation. Adult males and females were acclimated in water baths at 30°C or 34°C for 3 days. The temperature was increased by 1°C every 12 h until reaching the experimental temperatures. During the acclimation, the salinity, light: dark cycle, and dissolved oxygen concentration were kept at 30 PSU, 12 L:12D, and >5 mg L −1 by aerations, respectively (see also Truong et al., 2020). P. incicus were fed two times a day with Isochrysis galbana at 30,000-33,000 cell L −1 (~80 0-850 μg carbon per litter, Doan et al., 2018).

| Fish predator cue preparation
Barramundi larvae Lates calcarifer (15 individuals with a total length of 14 ± 1 mm) were reared in a 1-L bottle. Fish larvae were fed with ~100 P. incisus twice a day. After three rearing days, barramundi larvae were removed and the rearing water containing fish predator cues (FPC) was filtered through a 0.5 μm filter paper. This filtered water with FPC was divided into 50 ml aliquots and subsequently frozen at −20°C. FPC was thawed before being used in the experiment, as predator cue effects on the prey still remain after being frozen (Lürling & Scheffer, 2007).

| Transgenerational experiment
To test the transgenerational effect of MHW and FPC on the performance of the tropical copepod Pseudodiaptomus incisus, effects on two generations of P. incisus were studied (F1 and F2). F1 P. incisus were exposed to one of four treatments, including two thermal treatments (30 or 34°C) × 2 FPC (presence or absence) × 10 replicates ( Figure 1). The control temperature of 30°C was chosen as it is the mean coastal water temperature in the coastal water in southern Vietnam (see Appendix S1, Doan et al., 2018). We manipulated an experimental MHW condition with a temperature of 34°C, which is about ~2°C higher than the 90% temperature variations measured in the Cam Ranh Bay (Doan X.N., Pham, Q.H. and Dinh K.V., unpublished data).
To start the experiment, acclimatized F0 P. incisus carrying egg sacs (prosomal length = 797.43 ± 2.17 μm, clutch size = 16 ± 2 eggs) were assigned to 1.2-L plastic bottles (15 females each bottle) and In both generations, we analyzed clutch size (from fixed females carrying an egg sac), percentage of females with hatched eggs, and the number of hatched nauplii per clutch from alive females. Ten other males and females were used to test for survival of males and females, cumulative nauplii, and fecal pellets over 5 days. All data collection procedure was similar to our previous study .

| Cumulative nauplii and fecal pellets
To evaluate the reproductive output and the grazing rate of P. incisus, we quantified the cumulative nauplii and fecal pellets in 5 days.
Ten adults of both sexes were transferred to a separated 1-L bottle (10 replicates per treatment). Bottles were daily filtered using the same filtering net (see above) to collect the nauplii and fecal pellets. Alive adults were returned to the bottle while the dead ones were removed. The content containing nauplii and fecal pellets was transferred to a Petri dish and fixed with Lugol (4%). The number of nauplii and fecal pellets was counted using a stereomicroscope (SZ51, Olympus, Japan). In calanoid copepods, the fecal pellets were used as the index of the ingestion (Besiktepe & Dam, 2002), which typically has a positive correlation with egg production (Besiktepe & Dam, 2020).

| Statistical analyses
The data were analyzed using the R program ( quasi-distribution in the models, (iii) including random factors using generalized linear mixed-effects models (GLMER, package lme4 version 1.1-29), and/or (iv) changing to a negative binomial model using negative binomial generalized linear model (GLM.NB, package MASS version 7.3-57). The possible models were then validated using (i) Akaike information criterion (AIC) if available, (ii) plotting and assessing residuals versus fitted values, (iii) evaluating predicted versus actual values of response variables, and (iv) checking the normal distribution of the residuals. The best-fit model is the one that has the smallest AIC, minor errors in the predicted values, and more normally distributed residuals.

| DISCUSS ION
This is the first study investigating the role of transgenerational effects in shaping the individual and interactive effects of marine heatwaves in combination with non-consumptive predation stress on the tropical calanoid copepod P. incisus. We found a particularly strong effect of MHWs in the first generation, but the lethal MHW was fully ameliorated in the second generation. Interestingly, predation stress had little effect on the performance of P. incisus and played a minor role in shaping the transgenerational effects of MHWs on P. incisus. In the following paragraphs, we will first

| The effects of MHW (H1) and FPC (H2) and their interactions (H3)
Results in the F1 generation mostly confirmed and strengthened major findings of severe MHW impacts on P. incisus performance which were observed in our previous study focusing on

TA B L E 2
The results of statistical analyses testing the immediate and transgenerational effects of marine heatwave (MHW) and fish predator cues (FPC) on survival, reproductive parameters, and cumulative fecal pellets of F2 Pseudodiaptomus incisus.  .
Indeed, MHW exposure increased mortality, lowered hatching success, and cumulative nauplii and fecal pellets. Increased mortality likely occurred as a result of physiological dysfunctions, for example, the decline in ATP synthesis shows a tight correlation with copepod survival under thermal stress (Harada et al., 2019).
Two other general, but not exclusive, physiological mechanisms for the warming-induced mortality in ectotherms are the damage of macromolecules (Somero, 2010), and the higher cellular oxygen demand than the capacity of oxygen delivery (Pörtner, 2010).
The reduced performance of P. incisus was particularly strong for reproductive success with ~30%-60% reductions in the size of egg clutches, the percentage of females producing hatched eggs, hatched nauplii from a clutch, and cumulative nauplii -all widespread phenomena in tropical copepods under exposure to MHW Nguyen, Le, Doan, Pham, et al., 2020).
The lower reproductive outputs are generally related to reduced grazing (Besiktepe & Dam, 2020;Krause et al., 2017), thereby energy intake. While we observed a lower cumulative fecal pellets of MHW-exposed F1 P. incisus, the correlation of cumulative fecal pellets and nauplii was insignificant, suggesting that higher energy available for reproduction may be reduced due to higher energetic cost for basal maintenance under MHWs (Siegle et al., 2022).
The FPC presence caused a small increase in females with successfully hatched eggs, the number of nauplii hatched per clutch

TA B L E 2 (Continued)
F I G U R E 4 Immediate and transgenerational effects of the marine heatwave (MHW) and fish predator cues (FPC) on the survival (mean ± SE) of F2 Pseudodiaptomus incisus males (a) and females (b). *CT: Control temperature.
of P. incisus, which may be an adaptive response to predators. For example, predation can cause 50%-75% mortality in marine copepods, and increased reproductive outputs are a general mechanism to compensate for consumptive mortality (Hirst & Kiorboe, 2002).
Importantly, the FPC-induced increase in nauplii production only occurred at the control temperature but not under MHW exposure which suggests that copepods could not maintain high reproduction due to energetic cost (Low et al., 2018). Stronger MHW-induced reductions in cumulative nauplii and fecal pellets were observed in FPC-exposed P. incisus in our previous study , in which we could quantify both parameters for an entire adult lifespan.

| Parental effects of MHW (H4) and FPC (H5) on the F2 generation
The parental effects of MHWs and/or FPC occurred when P. incisus was only exposed to these stressors in the F1 generation but not in the F2 generation. One of our important findings was that parental exposure to MHWs (F1-MHW × F2-control) reduced reproductive success and grazing, but not survival of F2 P. incisus. The reduced F2 reproductive success and cumulative fecal pellets may result from reduced energy and resource investments for F1 reproduction, further limited by the lower F1 grazing. F1-MHW-induced poor maternal provisioning was likely the reason for the lower performance of F2 copepods. A similar result has been observed in a previous study on the same copepod species (Dinh et al., 2021).
Across treatments, the main parental effect of FPC accounted for only a ~2% positive effect on the cumulative nauplii and fecal pellets. The positive effect of parental exposure to FPC on the reproductive outputs of offspring is common in zooplankton and this effect may last for several generations after removing the predation stress (see, e.g., in Daphnia ambigua, Walsh et al., 2015). However, these positive parental FPC effects on P. incicus were at least an order of magnitude smaller than the negative parental MHW effects on these life-history traits and were unlikely to affect the overall sensitivity of P. incicus to MHW.

| Transgenerational plasticity of P. incisus to MHW (H6), FPC (H7), and their interaction (H8)
Transgenerational plasticity of P. incisus to MHW and/or PFC occurred when copepods were exposed to these stressors for both generations F1 and F2. There is mixed evidence of transgenerational effects of multiple stressors on the vulnerability of aquatic species.
On the one hand, there is evidence that corals (Torda et al., 2017) and coral fishes (Donelson et al., 2012) show rapid transgenerational acclimation to warming, and that can be linked to a complete compensation in aerobic scope (Donelson et al., 2012), the epigenetic changes (Ryu et al., 2018), or the associated microbes (Torda et al., 2017). On the other hand, there is evidence that transgenerational exposure to extreme warming, metal, fish predator cues , and pesticides    . The different degree of TGP reflects the nature of the stressors (mode of action, Schäfer & Piggott, 2018), the magnitude of the stress level (Donelson et al., 2012), the number of exposed generations ,

| Conclusions and perspectives
There is a great concern that the hyperdiverse tropical ecosystems may be collapsing under the cumulative impact of multiple stressors (Barlow et al., 2018;Dinh, 2019;Worm et al., 2006). Among others, MHWs are becoming a severe threat, increasingly causing mass and widespread mortality across taxa (Garrabou et al., 2009(Garrabou et al., , 2019 (Evans et al., 2020). Consequently, MHWs may substantially reduce the secondary biomass production of zooplankton which fuels resources and energy to the vast majority of marine predators such as corals, crustaceans, and fish (Arimitsu et al., 2021;Chew et al., 2012). Most importantly, our results suggest that P. incisus may evolve rapidly and complete TGP to MHW, with full amelioration of its lethal effect in the second generation. The transgenerational plasticity of MHWs comes with a cost of reduced reproduction, and grazing may be a crucial and widespread mechanism for marine invertebrates coping with the transient effects of heatwaves (Cavieres et al., 2020;Dinh et al., 2020;Doan et al., 2019). Interestingly, fish predator cues played a minor role in shaping both immediate and transgenerational effects of MHWs, highlighting the dominant effects of MHWs on P. incisus.
MHWs occur randomly across space and time and do not provide any reliable signal for long-term directional selection and evolution of thermal tolerance which may enable copepods to deal with the thermal stress relating to annual variations in temperatures across seasons, e.g., in temperate calanoid copepods (Sasaki & Dam, 2020). writing -original draft (equal); writing -review and editing (equal).

ACK N OWLED G M ENTS
This research was financially supported by the International Foundation for Science, Stockholm, Sweden, through a grant to Nam X. Doan (Grant I-2-A-6347-1). The constructive feedback of two anonymous reviewers considerably improved the manuscript. We thank Dr. Hoang Thi My Dung Le for statistical assistance and Dr.
Samuel J. Macaulay at the University of Oxford for proofreading and critical feedback on the revised manuscript.

CO N FLI C T O F I NTE R E S T
No conflict of interest to declare.